Enzyme Linked Immunosorbent Assay ( Elisa ) Lab Report

Enzymes are molecules that accelerate the rate of a reaction through the lowering of the activation energy necessary to perform the reaction without their presence. Depending on the environment that the enzyme is in, determines how efficient the enzyme will be in accelerating the reaction. Factors such as pH and temperature play a role on the enzyme’s efficiency and overall success of the reaction. For example, having a high temperature can break apart non-covalent interactions within proteins—the most common form of an enzyme. The breaking down of these bonds would result in the enzyme having a conformational change that does not allow the substrate to fit into its active site. In our experiment, we used the enzyme cellobiase in order to study

The purpose of this lab is to test for enzyme activity, look at enzyme specificity, and how temperature affects enzyme activity.

The ELISA test can also be used to detect antibodies that are produced in response to a specific antigen. Using information about how you completed this ELISA experiment, outline a procedure for testing for antibodies in the blood.

Colorimetric assay is a process of determining a concentration of a solution based on absorbance of light. The purpose of this lab is to determine if the Bradford assay is an accurate way to determine an unknown concentration of two samples of protein. The Bradford assay is done by measuring wavelength of light passing through a cuvette filled with Bradford dye and concentrations of PBS and proteins. After the cuvettes are mixed they are placed into a spectrophotometer to measure wavelength. The wavelength given will be used to plot a standard curve based on concentration (x-axis) and wavelength (y-axis). The standard curve is then used to measure an educated guess on the concentrations of unknown protein concentrations. We hypothesized that if we use the Bradford assay and colorimetric spectrophotometry we can determine an accurate concentration of two unknown concentrations of proteins. The results of this lab failed to reject our hypothesis based on accurate measurements of protein concentrations. The standard curves are drawn with a linear increasing slope. The Bradford assay is an accurate way to demine the concentration of an unknown concentration.

Enzymes are catalysts that function to speed up reactions; for example, the enzyme sucrose speeds up the hydrolysis of sucrose, which breaks down into glucose and fructose. They speed up reactions but are not consumed by the reaction that is taking place. The most important of the enzyme is the shape as it determines which type of reaction the enzyme speeds up. Enzymes work by passing/lowering and energy barrier and in doing so; they need to bind to substrates via the active. Once they do, the reaction speeds up so much more quickly than it would without the enzyme. Coenzymes and cofactors aid the enzyme when it comes to binding with the substrate. They change the shape of the active site so the substrate can bind properly and perform its function.

2. We measured 1 mL of turnip peroxidase (the enzyme) and 3 mL of neutral buffer (pH corresponding to the test tube number i.e. pH 5 in test tube 5) with a syringe and disposed it into tubes 3, 5, 6, 7, 8, and 10

Eisenthal, R. & M.J. Danson (Eds.) (2002). _Enzyme Assays: A Practical Approach_. United Kingdom: Oxford University Press

Gel electrophoresis method. Qualitative analysis shows protein concentrations in kidney, heart, and liver. 1-6 are kidney tissue. 7-14 are liver and 15-19 are the heart tissue.

After the substrate solution was added, five drops of the enzyme were quickly placed in tubes 3, 4 and 5. There were no drops of enzyme added in tubes 1 and 2 and in tube 6 ten drops were added. Once the enzyme solution has been added the tubes were then left to incubate for ten minutes and after five drops of DNSA solution were added to tubes 1 to 6. The tubes were then placed in a hot block at 80-90oC for five minutes. They were then taken out after the five minute period and using a 5 ml pipette, 5 ml of distilled water were added to the 6 tubes and mixed by inversion. Once everything was complete the 6 tubes were then taken to the Milton Roy Company Spectronic 21 and the absorbance of each tube was tested.

A clarified and physiologic buffered (pH 7 to 8) is added to the antibodies and purified ZA2GP to form a mixture that is added to the immobilized ligand.

Organisms cannot depend solely on spontaneous reactions for the production of materials because they occur slowly and are not responsive to the organism's needs (Martineau, Dean, et al, Laboratory Manual, 43). In order to speed up the reaction process, cells use enzymes as biological catalysts. Enzymes are able to speed up the reaction through lowering activation energy. Additionally, enzymes facilitate reactions without being consumed (manual,43). Each enzyme acts on a specific molecule or set of molecules referred to as the enzyme's substrate and the results of this reaction are called products (manual 43). As a result, enzymes promote a reaction so that substrates are converted into products on a faster pace (manual 43). Most enzymes are proteins whose structure is determined by its sequence of its amino acids. Enzymes are designed to function the best under physiological conditions of PH and temperature. Any change of these variables that change the conformation of the enzyme will destroy or enhance enzyme activity(manual, 43).

Guidelines are important in all fields, but especially so in the field of biomedical science and engineering. There is a great deal of pressure on scientists to solve the ailments that plague our society, and one of the methods to accomplish this is immunotherapy. Immunotherapy can have a huge impact on how we treat diseases such as cancer or even just basic bacterial infections. The study of antibodies can also assist those who are immunocompromised, such as HIV/AIDS patients, or those who have other immunodeficiencies, such as autoimmune disorders or those who have recently undergone organ transplants. With so much riding on the study of these small but important proteins, it is so important to have the right materials and knowledge of how to use such materials for accurate results.

Western blotting — Western blotting was performed as described previously (2). Primary antibody against PPARδ was from Cayman Chemical. The secondary antibody IRDye-800CW (925-32211) and IRDye streptavidin (925-68079), IRDye Maleimide (929-80020) were from LI-COR. The infrared signal was detected using an Odyssey Infrared Imager.

Protein A is thermostable, and is not destroyed by trypsin4; however, Protein A is most useful for its stability in regard to pH. It’s polypeptide structure is so stable that it has continuous stability both at very acidic pH (0.99) and at a very basic pH (11.8) 3,4. For this reason, it useful across many pH levels that would be useful in a protein affinity assay because this particular assay requires changing the pH to determine what would be the best to elute the protein that is being selected for; however, for an affinity assay involving human immunoglobulin, protein A is used for an additional reason. Protein A is from a bacteria and human immunoglobulin is associated with the immune system of the humans, as the name implies. Protein A contains five locations that it can bind to IgG5. These five domains, often labeled regions A-E, each consist of 58-62 amino acid residues, and a C-terminal consists of 150 amino acid residues. The IgG binding domains (A-E) each consist of three anti-parallel α-helices which are stabilized by hydrophobic regions between them. These domains bind with the Fc region of the immunoglobulin and form a complex with the Fc region. When this binding occurs a conformational change occurs which is reinforced by polar, and hydrophobic interactions4. This binding pattern is the basis for its use in protein affinity assay that involve eluting IgG out of solution that contain other human immunoglobulins. As the other

The last part of this procedure, antibodies will be used to detect one specific protein from others on the membrane. Incubate the membrane with 10 ml of primary antibody for 10-20 minutes and then place on a rocking platform. Rinse the membrane quickly after with wash buffer on a rocking platform. After three minutes is up, discard the wash and incubate the membrane with 10 ml of secondary antibody for 5-15 minutes on the platform. Rinse the membrane again shortly after and wash the membrane for three minutes. Next, discard the wash and add 10 mL of HRP color detection reagent. Incubation will occur after this for 10-30 minutes. Once it is done, rinse the membrane twice with distilled water and blot dry.